Molecular diagnostics and inhibition of cross‐reactive carbohydrate determinants in Hymenoptera venom allergy

Abstract Background The composition of venom extracts, cross‐reactive carbohydrate determinants (CCD) and the component‐resolved diagnostics (CRD) are important fields of investigation. IgE‐reactivity to CCD complicates the interpretation of IgE to Hymenoptera venoms, especially in patients with multiple‐positivity. We analyzed the clinical importance of CRD and CCD‐inhibition for selection of allergens for venom immunotherapy (VIT). Methods In 71 patients, we measured specific IgE (sIgE) to honeybee venom (HBV), wasp venom (WV), hornet venom (HV), CCD, and recombinant allergens: phospholipase A2 (rApi m 1), hyaluronidase (rApi m 2), icarapin (rApi m 10), antigen 5 (rVes v 5), and phospholipase A1 (Immunoblot). In 29/71 HBV/WV/HV/CCD‐positive patients CCD‐inhibition was performed. According to CRD and CCD‐inhibition, we identified true sensitization and defined groups of multiple‐positive patients who needed CCD‐inhibition before starting VIT. Results sIgE‐rApi m 1, sIgE‐rApi m 2, and sIgE‐rApi m 10 were detected in 65.7%, 68.4%, and 58%, respectively. In HBV allergic patients, CRD sensitivity was 86.8%. In WV allergic patients, sensitivity of sIgE‐rVes v 5 was 94%. True multiple‐sensitization was found in 44.8% of HBV/WV/HV/CCD‐positive patients after CCD‐inhibition. Patients with multiple venom‐ and CCD‐positivity had more frequent severe allergic reactions (p < 0.001). CCD‐inhibition was helpful in HBV/WV/HV/CCD‐positive patients who were negative to all tested recombinant honeybee allergens. Persistence of HBV‐positivity after CCD‐inhibition requires CRD to other honeybee recombinant allergens. Conclusion CRD, using a profile of five most important recombinant allergens and CCD, has a high sensitivity for the diagnosis of venom allergy, especially in patients positive to several venom extracts. CRD and CCD‐inhibition are helpful to reveal the clinically relevant, true sensitization and improve the selection of venoms for long‐lasting VIT.


| BACKGROUND
Hymenoptera venoms are known to cause life-threatening and fatal immunoglobulin E (IgE)-mediated anaphylactic reactions. About 50%-60% of patients with Hymenoptera venom allergy (HVA) have specific IgE (sIgE) reactivity to both insect-species, honeybee (Apis mellifera) and yellow jacket (Vespula vulgaris, also called wasp) or hornet (Vespa crabro). Protein-based and carbohydrate determinantsbased cross-reactivity are the most frequent causes of multiplepositivity. 1 Venom extracts are complex, heterogeneous mixtures rich in glycoproteins. Cross-reactive carbohydrate determinants (CCD), composed of α-1,3-fucosylated N-glycans, interfere with the detection of clinically relevant IgE specific to protein epitopes. 2 In contrast to diagnostics based on whole venom extracts, the component-resolved diagnostics (CRD) reveal sIgE to single recombinant allergens without CCD. In patients positive to several venom extracts, CRD can distinguish true, clinically relevant IgE sensitization to protein epitopes from sIgE-reactivity to CCD. Compared to diagnostics based on whole venom extracts, CRD improves the selection of allergens that will be used for long-lasting venom immunotherapy (VIT). [3][4][5] Hymenoptera proteins involved in cross-reactivity are hyaluronidases (Api m 2 and Ves v 2) and dipeptidyl-peptidases (Api m 5 and Ves v 3) from honeybee and wasp, respectively. 2 Cross-reactivity caused by CCD can be eliminated using CRD and CCD-inhibition that are underused in clinical practice. 5 False-positive results are a common problem in the in vitro diagnostics of HVA. 6 The majority of venom allergens are glycoproteins and cross-linking of only CCD epitopes with mast cell-bound IgE does not lead to mast cell degranulation. sIgE-reactivity to whole venom extract does not allow a distinction between sIgE-reactivity to protein epitopes from sIgEreactivity to CCD epitopes. 7,8 Extract-based diagnostics limit the use of standard sIgE serology for selection of causative venom for VIT. In some cases, despite finding of sIgE, patients did not have allergy symptoms, because sensitization was caused by well-tolerated venom allergen. VIT over 3-5 years with insect venom is still the only effective and causal treatment of HVA. The clinical importance of sIgE against CCD epitopes (CCD-sIgE) is controversial, although most studies showed that CCD-sIgE have no clinical relevance. [9][10][11] Due to the clinically irrelevant CCD-sIgE, increased levels of sIgE to conventional whole venom extract should be interpreted carefully, in the context of the clinical history. Unfortunately, identification of the allergy-relevant venom is sometimes difficult, because the patient could not identify the insect. 4 Moreover, some patients with high sIgE-levels do not show clinical manifestations, while other patients with low sIgE-levels may experience life-threatening systemic allergic reactions (SAR). 4,8 In our previous study, we showed that CCD-sIgE were more frequently found in honeybee allergic patients. 4 This finding can be explained by the fact that majority of honeybee allergens are glycosylated, while the two major wasp allergens are not glycosylated. 12,13 Also, we found that the use of the major allergen rApi m 1 (phospholipase A2) was not sufficient, regardless of the detection method. On the other hand, using recombinant major wasp allergen rVes v 5 (antigen 5) was enough for about 90% of wasp allergic patients. 4 The variability of test systems in allergy diagnostics is due to a lack of international standards for allergens and antibodies. This often leads to different test results and complicates their comparability.
This study aimed to analyze the diagnostic importance of CRD for simultaneous determination of five most important recombinant allergens and CCD-inhibition in patients with HVA. According to the reliable history of insect sting allergy, and the sIgE-test we intended to identify criteria for true sensitization and to define groups of multiple-positive patients who needed CCD-inhibition before starting long-term VIT.

| Statistical analyses
Obtained data were analyzed using IBM SPSS Statistics software for Windows (version 17; IBM, Armonk, NY). Mean quantitative variables were used and frequency of qualitative variables were also calculated. Nonparametric Chi-square and Fisher exact tests were used to evaluate the relationship between variables. The Wilcoxon signed-rank test and t-test were used to compare two matched samples. According to a history of insect sting allergy, we calculated sensitivity using the formula: Sensitivity = true-positive/true-positive + false-negative � 100%.
The majority of patients (55/71) had severe SAR (III and IV grades of SAR) (77.5%) (p < 0.001). No significant differences were found in the frequency of severe SAR between female and male patients.
In the CCD-sIgE positive group, more patients had severe SAR to Hymenoptera sting 27/29 (93%) than mild SAR 2/29 (6.9%) All six hornet allergic patients had sIgE-reactivity to rVes v 5, while 2/6 (33.3%) of them had sIgE-reactivity to rVes v 5 and rVes v 1. There were no wasp or hornet allergic patients who had an isolated sIgE-reactivity to rVes v 1 (Tables 2 and 3). patients were negative for all tested recombinant allergens (Tables 2   and 3).
According to CRD and CCD-inhibition, five different subgroups of HBV/WV/HV/CCD-positive patients were distinguished (Table 3).
In the first group of selected patients (7/29) with a history of SAR to honeybee and wasp/hornet stings (except for one patient who T A B L E 1 Comparison of demographic characteristics and degree of severity systemic allergic reaction in 71 patients with Hymenoptera venom allergy, with and without CCD-sIgE.

CCD-sIgE positive (n = 29)
Age (years) 43  The second group (4/29) of patients with a history of SAR due to wasp/hornet sting had sIgE-reactivity to rApi m 2 and rVes v 5, before and after CCD-inhibition.
In the third group of selected patients (9/29) with a history of SAR to honeybee sting, IgE-testing confirmed a honeybee allergy due to sIgE-reactivity to the majority of tested recombinant honeybee allergens and loss of WV-positivity (p < 0.01) and HV-positivity (p < 0.05) after CCD-inhibition. We showed that CCD-inhibition significantly increased sensitivity for HBV-single-positivity from 0% to 77%.
In the fourth group of selected patients (3/29) with a history of SAR to honeybee sting, there was no sIgE-reactivity to all tested recombinant allergens. After CCD-inhibition, in 2/3 patients we found sIgE-reactivity to rApi m 1 or rApi m 2.
The fifth group of selected patients (6/29) with a history of SAR to wasp/hornet stings, showed initial sIgE-reactivity only to rVes v 5.
In the group of 29 HBV/WV/HV/CCD-positive patients, all patients completely lost CCD-positivity after CCD-inhibition (p < 0.01).
22/29 patients retained HBV/WV-or HBV/WV/HV-positivity, but a significant decrease was found in the mean concentration of sIgE to WV and HV extracts (Table 4). A significant decrease was found in the mean concentration of sIgE to rVes v 5. On the contrary, a substantial increase was found in the mean concentration of sIgE to rApi m 1 and rApi m 2 after CCD-inhibition (Table 4).
According to our results, we proposed an algorithm for the necessary diagnostic steps to ensure an adequate therapeutic approach for long-lasting VIT ( Figure 2).

| DISCUSSION
Our study demonstrates the clinically importance of molecular CRD, Our previously published study showed that we must increase the number of recombinant honeybee allergens, tested by Immunoblot. 4 We especially analyzed a group of patients with multiplepositivity in order to determine how CCD-inhibition affects sIgEresults and whether it is necessary to apply CCD-inhibition in all CCD-sIgE-positive patients.
We found that 39/71 patients had multiple-positivity, while 29/ 39 patients had CCD-sIgE. More recent studies revealed that much of the cross-reactivity between wasp and honeybee allergens is not due to protein cross-reactivity but is caused by CCD. 16 to epitope availability, immunoreactivity, and enzymatic function of native allergens. 8,22,24 CCD-sIgE have been reported to be responsible for more than 50% of double-sensitizations to HBV and WV. 25,26 Both, previously studied data and our data showed that about half of the patients with HVA develop CCD-sIgE. 4,26 CCD-sIgE represent a pitfall of in vitro diagnostics because they cause multiple-reactivity with any glycosylated allergens (pollen, food, or venom) and significantly interfere with the detection of clinically relevant IgE-sensitization to protein epitopes. 5,27 The double-or triple-positivity of sIgE to whole venom complicates the choice of appropriate VIT, resulting in increased risk of side effects, de novo sensitizations and higher costs. 19 In these patients, CRD and CCD-inhibition provide more accurate diagnostic results. 7,17 In most countries, the accepted clinical standards recommend skin-testing as the initial method. 28 However, skin-testing may show In this context, the newly detected sIgE-reactivity to recombinant honeybee allergens in our four venom-allergic patients after CCD-inhibition was very important. It is already known, that sIgElevels do not correlate with the severity of the allergic reaction. 4,33 Also, long-lasting VIT should be considered for patients with honeybee sensitization because they are at a higher risk for SAR. 32 Surprisingly, we are the first to observe a significant increase of   This leads to false, higher sIgE-levels to rVes v 5 before CCDinhibition. CCD-inhibition reduces non-specific binding of CCD-sIgE leading to true, lower sIgE-levels to rVes v 5.
Our results indicated that CCD-inhibition can overcome current limitations of some procedures in recombinant technology, especially in Immunoblot which do not display three-dimensional conformational structure of antigens that hide or change presentation of some IgE epitopes. 37 Nevertheless, the recombinant production of certain indicate VIT-induced improvements in immune tolerance. 19,43 Since the IgG/IgE ratio is a good marker of efficacy, 44 it is important to know their true initial concentrations, before starting long-term VIT.
Future studies will elucidate whether sIgE-levels before or after CCD-inhibition should be taken as clinically relevant.
Our results demonstrate that the CCD-inhibition test is useful in patients who tested negative for recombinant honeybee allergens.
Our patients, who had persistent HBV-positivity even after CCDinhibition, require CRD to other recombinant honeybee allergens.
Molecular diagnostics is a major step toward personalized medicine, 45 especially in patients who are exclusively sensitized to allergens that are underrepresented in therapeutic mixtures for VIT, as described for rApi m 10. 46 We demonstrated that Immunoblot,